This application claims priority to Japanese Patent Application Number 11-56,960, filed Mar. 4, 1999, and to Japanese Patent Application Number 11-58,434, filed Mar. 5, 1999.
1. Field of the Invention
This invention relates to an anti-viral agent useful for treatment of infectious disease caused by human immunodeficiency viruses (HIV). In particular, it relates to a polypeptide which can inhibit HIV-1 infectious disease, a DNA coding for said polypeptide, and a process for producing said polypeptide.
2. Description of the Related Art
Currently, reverse transcriptase inhibitors and protease inhibitors are used for medical treatment of acquired immune deficiency syndrome (AIDS) caused by HIV-1 virus. As for the reverse transcriptase inhibitors, nucleoside type compounds such as zidovudine (AZT, 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxycyltidine), didanosine (ddI, 2xe2x80x2,3xe2x80x2-dideoxyinosine), zalcitabin (ddC, 2xe2x80x2,3xe2x80x2-dideoxycytidine), sutavudine (d4T, 2xe2x80x2,3xe2x80x2-dideoxy-2xe2x80x2,3xe2x80x2-didehydrothymidine) and lamivudine (3TC,3xe2x80x2-thiacytidine), or non-nucleoside compounds such as nevirapine, are used.
Concerning nucleoside type reverse transcriptase inhibitors, it is known that resistant strains will appear within about one year after administration of the inhibitors and some serious side effects are caused by chronic administration. On the other hand, side effects are rarely caused by administration of non-nucleoside type reverse transcriptase inhibitors, resistant strains tend to appear in early stages of an administration, because of high specificity of these inhibitors. Nowadays, saquinavir, ritonavir, indinavir and the like are used for protease inhibitors. Although these protease inhibitors exhibit considerable anti-viral activity by single administration, in general the effect remains to be only transient. The sensitivity of HIV strains tends to decrease by mutation of the amino acid sequence of HIV proteases. In addition, there are problems in respect to stability in living body and occurrence of side effects such as disorder on digestive organ.
Recently, the U.S. AIDS Clinical Trials Group reported that the combination therapy using AZT and ddI, or AZT and ddC brought better results in three indexes described below, compared with the monotherapy using AZT alone, which have been recognized to be the first choice until now. The indexes are (1) life-extending effect, (2) the prevention to AIDS onset, and (3) reduction on number of cluster of differentiation (CD) 4 positive cells. Moreover, results on combination therapy between reverse transcriptase and a protease inhibitor were reported in succession. When a protease inhibitor was used in combination with two reverse transcriptase inhibitors (for example, AZT+ddI or AZT+3TC), it was found out that any protease inhibitor exhibited potent anti-viral activity. That is, the amount of viruses in blood was reduced to less than the detection limit in 60-90% of cases.
Thus, it is suggested that the amount of virus in the body might be reduced to a very low level by combination of plural anti-HIV agents, which effect at difference points through different mechanisms of action, and consequently, the onset of the disease could be prevented. However, the problems, such as the appearance of HIV-1 strains with multiple drug resistance and occurrence of some side effects by chronic administration, still remain to be solved. Moreover, although the proliferation of the virus is inhibited by the concomitant drug therapy, the viruses infected to the cells do not vanish completely. Recently, it was confirmed that HIV is produced in large quantities in a living body even under a prolonged latency period and the body repeats the immunologic elimination of the HIV, which leads to the aspect of the dynamic balance between the virus and the host. Therefore, in order to prevent the onset of AIDS, it is important to inhibit proliferation of the virus in the latency period and to suppress the level of the virus in blood to a low amount. Accordingly, in addition to the reverse transcriptase inhibitors and the protease inhibitors, development of an anti-HIV agent which acts through other mechanisms than conventional anti-HIV agents is desired. In concrete, such anti-HIV agent should act at its target other than the reverse transcriptases and the proteases utilized in the life cycles of HIV including an initial process of HIV infection.
HIV infection occurs by attachment of HIV to a host cell succeeded by entry of HIV into the host cell. The adhesion occurs through binding of glycoprotein120 (gp120), HIV envelop glycoprotein, and CD4, a receptor existing on the surface of the host cell. Therefore, a compound which inhibits binding of gp120 and CD4 is expected to inhibit attachment of HIV to the host cell, which enables protection from HIV infection. Then, an attempt to inhibit binding of gp120 and CD4 has been performed. For example, soluble CD4, prepared by genetic engineering technique, was administrated to the body on trial. Though inhibition activity was observed in vitro by the method described above, anti-HIV activity was not observed in vivo because of its short half life period. Moreover, it was revealed that infection did not occur when human CD4 was expressed in a mice cell, indicating existence of the second attachment molecule.
Recently, a series of results were reported that this second receptors belong to family of chemokine. HIV strains are divided roughly into two groups, one group contains strains exhibiting macrophage-tropic (M-tropic HIV) and another group contains strains exhibiting T cell-tropic (T-tropic HIV). It was suggested that the cell tropism observed between these virus strains is based on the difference in the molecular species of the second receptor. That is, it became clear that the type of HIV strains was determined by the type of the second receptor expressed on the target cells, namely CC chemokine receptor 5 (CCR5: M-tropic HIV receptor) or CXC chemokine receptor 4 (CXCR4: T-tropic HIV receptor). From these new knowledge, the manner of HIV adopted to invade into the target is hypothesized as follows at present. First, gp120 binds to CD4 and then the conjugate binds to CCR5 or CXCR4 expressed on the host cells. As the result, the structure of gp120 alters with denuding of glycoprotein41 (gp41). Then, it adheres and invades into a cell membrane which leads to formation of a giant cell (syncytium) and results in occurrence of infection. It was found that addition of the corresponding chemokine inhibited infection of HIV by competitive blockade of binding of HIV to the chemokine receptor on the cells. This series of discovery not only accelerated the elucidation on the mechanism of infection and onset of HIV, but also provided a novel viewpoint concerning anti-HIV strategy.
To obtain a novel anti-HIV medicine which affects to such mechanism, the inventors have tried to obtain a compound that inhibits syncytium formation caused by fusion of HeLa cells expressing envelope glycoprotein (gp120 gp41) and HeLa cells expressing CD4 and CXCR4. The inventors have searched such compound in metabolic products of microorganisms. As the result, excellent HIV-inhibitory activity was found in the culture fluid of strain K97-0003, a newly isolated strain from soil. The strain is deposited to National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (1-1-3, Higashi, Tsukuba-shi, Ibaragi-ken, 305-8566, Japan) on Dec. 9, 1998, as strain K97-0003 (FERM BP-6670).
In the following, this invention is explained in detail. However, the detailed explanation of these preferred embodiments and examples are not intended to limit or restrict the range of the present invention.